I am doing Exps on autophagy mechanisms.
A question is that: when to add autophagy treatment (CQ, 3-MA, Rapa) into cultre models?
Immediately when seeding? Or after the cells adhesion into a monolayer?
thx
It depends on the type of the cells that you use. If the cells require adhesion for the normal growing ( like epithelial or vascular cells) than you need to wait till cell adhere to the bottom of the flask (overnight or at least for 4 hours) and start your treatment afterwards.
Majority of cells are going through the stress after splitting (trypsinization), so you definitely need to wait for at least a couple of hours if you use adherent cells ( I do not know which ones you use so they might need longer time to adhere). Brain cells require very gentle handling. So overnight waiting period is better for them. I guess that you might need to test the time/ use time-dependent experiment to avoid confounding factors with your specific cells. You do not really what to increase the effect of the agents because the cell membranes were damaged by trypsin. You need to check the cell density as well. If cells are overconfluent that might decrease the cell surface available for the agent to penetrate.
It is different if you use blood cells/leukemia cells that cannot really adhere and they do not need to adhere for normal functioning. Regards
Thanks to Olga Sukocheva.
Actually I use huh7.5.1, hepG2, AD38 cells. All of them are adherent cells.
What I am planning to use:
CQ 50 uM, 100 uM
3-MA 5 mM, 10 mM
Rapa 100 nM, 300 nM
And the preliminary time test would be 6 hrs.
Do you, or somebody else with experience can show me that whether the above protocol is appropriate?
thx
first, some cells are very sensitive to these inhibitors and you need to optimize your conditions. The second point, if you like to stop autophagy process after inducing it to compare whether it is a cell death or cell survival you may add it just hours (2-3) before treatment. However, many articles prefer to use it as pre-treatment 1 hour before any other treatment.
The 6h as the first time point is ok as it was published previously ( See the attached paper for HepG2 cells). You need to do time-course; 6 h 12, 24, 48 etc. However, in case of cancer cells it takes days to see the really good response. It took 7 days with 100 nM rapa for me to see the statistically significant response in breast cancer cells. So, please expect that you might need to do longer treatment if there is no differences with the 6-12h treatments. There is also a difference between batches of the same cells. So, do not get upset if you do not see responses. Just increase the dose or time of treatment. Depends on your batch of cells, you might see the strong response at 6h, however, I think it is an unlikely scenario. Regards
- Badges
- Science topic